WO2023286012A1 - Collagen powder, process for its preparation and uses thereof - Google Patents
Collagen powder, process for its preparation and uses thereof Download PDFInfo
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- WO2023286012A1 WO2023286012A1 PCT/IB2022/056508 IB2022056508W WO2023286012A1 WO 2023286012 A1 WO2023286012 A1 WO 2023286012A1 IB 2022056508 W IB2022056508 W IB 2022056508W WO 2023286012 A1 WO2023286012 A1 WO 2023286012A1
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- Prior art keywords
- collagen
- test
- collagen powder
- powder according
- animals
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/41—Particular ingredients further characterized by their size
- A61K2800/412—Microsized, i.e. having sizes between 0.1 and 100 microns
Definitions
- Collagen is the predominant protein in the connective tissue of animals, including humans, and is the most abundant protein in mammals (about 25% of total protein mass), constituting about 6% of body weight in humans, being found in all supporting structures such as ligaments, muscles, joints, synovial membranes, cartilage, bone, and skin. Collagen is composed of fibers oriented parallel to each other so as to guarantee good mechanical support as well as high tensile strength.
- Type I collagen accounts for 90% of total collagen and is involved in the composition of the major connective tissues, such as skin, tendons, bones and cornea.
- the structural unit of collagen is represented by tropocollagen, which is a protein of about 285 kDa formed by three polypeptide chains with a left-handed course that are combined to form a right-handed triple helix.
- tropocollagen is a protein of about 285 kDa formed by three polypeptide chains with a left-handed course that are combined to form a right-handed triple helix.
- Synovial membranes and tendons become fragile, less tensile-strength resistant, and cartilage becomes thinner or even completely disappears.
- Collagen administrations and infiltrations can slow this process and, in some cases, accelerate post-injury regeneration.
- Collagen infiltrations are applied in case of muscle injuries and injuries of the joint ligament system (e.g., muscle strains or ligament sprains), in joint, vertebra and tendon degeneration processes, e.g., in tendinitis.
- Various supplements, creams and powders containing collagen are commercially available for a variety of uses, from pharmaceutical use to food use. However, there is still a need for providing new collagen-based formulations that are versatile and allow the use in a wide range of applications.
- Objects of the invention It is an object of the invention to provide a new hydrolyzed collagen powder having a specific particle size.
- FIG 2 shows the experimental numerical data of the particle size distribution in Figure 1.
- Figure 3 shows the structured approach to select a sample preparation method [Excerpt from ISO 10993-3: 2014].
- subject-matter of the invention is a hydrolyzed collagen powder (hereinafter also just "collagen powder") having the following particle size distribution: - at most 10% of the particles have a mean diameter (d0.1) lower than 2.5 microns; - at least 50% of the particles have a mean diameter (d0.5) lower than 10 microns; - at least 90% of the particles have a mean diameter (d0.9) lower than 20 microns.
- collagen powder hereinafter also just “collagen powder” having the following particle size distribution: - at most 10% of the particles have a mean diameter (d0.1) lower than 2.5 microns; - at least 50% of the particles have a mean diameter (d0.5) lower than 10 microns; - at least 90% of the particles have a mean diameter (d0.9) lower than 20 microns.
- Mean diameter herein means the mean diameter of the single collagen particle obtained by the process described below, whose shape is, to a first approximation, comparable to a sphere. According to a preferred embodiment, in the collagen powder of the invention, 100% of the particles have a mean diameter from 0.5 to 50 microns, preferably 0.6 to 40 microns.
- the collagen oligopeptides are characterized by the following molecular weights: - at least 30% of the oligopeptide molecules have a weight average molecular weight from 80 to 120 kDa; - at least 25% of the oligopeptide molecules have a weight average molecular weight from 50 to 70 kDa; - at least 25% of the oligopeptide molecules have a weight average molecular weight from 25 to 45 kDa; - at least 85% of said collagen has a molecular weight from 25 to 120 kDa.
- the collagen oligopeptides are characterized by the following molecular weights: - at least 30% of the oligopeptide molecules have a weight average molecular weight from 80 to 120 kDa; - at least 25% of the oligopeptide molecules have a weight average molecular weight from 50 to 70 kDa; - at least 25% of the oligopeptide molecules have a weight average molecular weight from 25 to 45 kDa; - at least 14.5% of the oligopeptide molecules have a weight average molecular weight from 100 to 110 kDa; - at least 11.5% of the oligopeptide molecules have a weight average molecular weight from 80 to 90 kDa; - at least 8.0% of the oligopeptide molecules have a weight average molecular weight from 40 to 45kDa; - at least 85% of said collagen has a molecular weight from 25 to 120 kDa.
- the collagen oligopeptides are characterized by the following molecular weights: - at least 3.0% of the oligopeptide molecules have a weight average molecular weight of about 120 kDa; - at least 14.5% of the oligopeptide molecules have a weight average molecular weight from 100 to 110 kDa; - at least 11.5% of the oligopeptide molecules have a weight average molecular weight from 80 to 90 kDa; - at least 7.0% of the oligopeptide molecules have a weight average molecular weight of about 70 kDa; - at least 8.0% of the oligopeptide molecules have a weight average molecular weight of about 60 kDa; - at least 10.5% of the oligopeptide molecules have a weight average molecular weight of about 50 kDa; - at least 8.0% of the oligopeptide molecules have a weight average molecular weight from 40
- the hydrolyzed collagen powder of the invention is produced by proteolysis from atelocollagen, which in turn is derived from the removal of the telopeptide terminals of the type I collagen.
- the collagen of the invention is of equine origin.
- the equine biological tissue the collagen is extracted from is the tendon, particularly the flexor tendon.
- the collagen powder of the invention shows the particle size distribution of Figure 1.
- subject-matter of the invention is a process for preparing the collagen powder of the invention that comprises: (i) suspending collagen of type I, deprived of the telopeptide fractions, in acidified water and subjecting it to proteolysis; and (ii) drying said collagen in a micronized form.
- step (i) concentrated acid suspensions of collagen having weight concentrations from 3% to 5% (w/v) collagen, are incubated with proteolytic enzymes, such as pepsin, in order to modulate the molecular weights of the collagen polypeptide chains.
- proteolytic enzymes such as pepsin
- the suspension is sprayed in the form of aerosol, which is then rapidly dried so as to obtain a powder; this process is known in industrial practice as "spray-drying.”
- the proteolysis can be carried out by using an acid solution, such as acetic acid in demineralized water, at pH between 3.0 and 4.0, containing 1.0- 10.0%, w/w pepsin (wt pepsin/wt collagen) at a temperature of about 37°C, for a few hours, such as 2-24 hours.
- Other methods can alternatively be used to produce the collagen powder of the invention.
- the collagen powder of the invention has proven to be particularly effective in a variety of uses, such as for example as a supplement, in cosmetics and in the medical field.
- the collagen powder of the invention can be used as it is or, if necessary, after appropriate sterilization treatment (e.g., by using ⁇ or ⁇ irradiation, according to known techniques).
- the collagen powder of the invention can be sprayed or applied to wounds, burns, sores or the like, or alternatively it can be formulated into a pharmaceutical or nutraceutical or cosmetic composition, or included in medical devices.
- “Pharmaceutical, nutraceutical or cosmetic composition” means a composition comprising the collagen powder of the invention in association with at least one carrier acceptable for the use such composition is intended for.
- a “pharmaceutically acceptable carrier” includes diluents, preservative agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents, and dispensing agents. Descriptions of pharmaceutically acceptable carriers and factors involved in their selection are known in the art. Depending on the use the composition of the invention is intended for, it may be liquid or solid, or may be formulated into gels, creams, ointments, pastes and the like, which may also be sterile, if required, such as in the case of injectable preparations or preparations to be used on open wounds.
- the collagen powder of the invention can also be used in combination with medical devices, such as, but not limited to, medicated patches and gauzes for the treatment of wounds, burns and sores of any nature, or it can be packaged as sterile solution in pre-filled syringes.
- medical devices such as, but not limited to, medicated patches and gauzes for the treatment of wounds, burns and sores of any nature, or it can be packaged as sterile solution in pre-filled syringes.
- the use of the collagen powder of the invention, or compositions containing it, in therapy, in cosmetics and as dietary supplement constitutes further subject-matter of the invention.
- the collagen powder of the invention can be used for the treatment of wounds and sores, by direct application or through medical devices, or it can be used for the treatment of joint disorders, such as by infiltration, for the treatment of interstitial cystitis, such as by injection of viscous and/or adhesive compositions, for vaginal application, such as in the treatment of vulvovaginitis, etc.
- the collagen powder of the invention can also be used for the regeneration and bio- revitalization of dermis and for the visco-supplementation and regeneration in the synovia in cases of osteo-chondral damage.
- the collagen powder of the invention is very useful for preventing and treating skin aging and can be administered in form of creams, lotions, gels, and the like or injected under the skin as filler, for example, but not limited to, for wrinkle smoothing or for solving other aesthetic problems.
- the collagen powder of the invention can also be taken orally as a dietary supplement or be included in cosmetic compositions.
- composition of the invention may comprise, in addition to the powder of the invention and any appropriate carriers and additives, other active ingredients, such as hyaluronic acid or salts thereof, e.g., sodium hyaluronate, of various molecular weights, chondroitin or salts thereof, e.g., chondroitin sulfate, probiotics, prebiotics, postbiotics, vitamins, such as Vitamin C, antimicrobial agents such as silver, and the like.
- active ingredients such as hyaluronic acid or salts thereof, e.g., sodium hyaluronate, of various molecular weights, chondroitin or salts thereof, e.g., chondroitin sulfate, probiotics, prebiotics, postbiotics, vitamins, such as Vitamin C, antimicrobial agents such as silver, and the like.
- the subject-matter of the invention is a method to treat sores, burns or wounds, joint disorders, aging skin, interstitial cystitis and vulvovaginitis, which includes administering an effective amount of the powder or composition of the invention to an individual in need thereof.
- the individual receiving the powder or composition of the invention is generally a mammal, including, but not limited to, human beings.
- Protein standards with precise molecular weights (markers) between 10 and 250 kDa were also charged in order to correctly determine the molecular weights of the proteins with unknown molecular weights.
- the run was conducted at 70 V for about 10 minutes in the concentration gel and at 120 V for about 2 hours in the separation gel.
- the gel was immersed in Coomassie-based fixation solution (0.125% Coomassie Blue R 250, 40% methanol, 10% acetic acid) for 1 hour.
- the gel was immersed in a decolorizing solution (40% methanol, 10% acetic acid) overnight at 4°C and captured.
- Example 2 An injectable solution is prepared in pre-filled syringes comprising, for each 50 ml syringe, 80 mg of collagen powder from Example 1, water/saline solution, hyaluronic acid, chondroitin sulfate, buffers q.s. to pH.4.5
- Example 3 A medicated gauze comprising 20 mg of collagen powder from Example 1 is prepared, integrated in a 2%/gr/25cmq aqueous solution of glycerin.
- Example 4 A mixture of 100 mg of collagen powder from Example1, added with rice starch or cornstarch, a mixture of triglycerides, isopropanol and medical petroleum, is prepared to make a spray formulation - 10 g content in 100 ml spray cans.
- Example 5 A high-porosity medical silicone base is prepared, integrated with a thin layer of collagen - foil - charged with 100 mg of collagen powder from Example 1 for an area of 100 sq. cm.
- Example 6 A solution of product is prepared for vaginal irrigation in ampoules containing, for each 10 mL ampoule, 10 mg of collagen powder from Example 1, water/saline solution and, depending on the desired recommendations for use, one or more components selected from probiotics, postbiotics and prebiotics; a buffer solution q.s. to pH 4.5 is added to the resulting composition.
- Example 7 A solution of product is prepared in vaginal ovules containing 5 mg of Example 1 collagen powder, water, glycerol, lactic acid, and depending on the desired recommendation for use, one or more components selected from probiotics, postbiotics and prebiotics.
- Example 8 A vial is prepared with 80 mg of the powder from Example 1 to be used for the regeneration/bio-revitalization of dermis. The powder should be reconstituted with WFI before use.
- Example 9 A vial is prepared with 80 mg of the powder from Example 1 to be used for the visco- supplementation and regeneration in the synovia in cases of osteo-chondral damage. The powder should be reconstituted with WFI before use.
- EXPERIMENTAL TESTS The collagen of the invention was subjected to several experimental tests.
- BIO ACTIVE COLL collagen Type I powder, obtained by the process of the invention, Example 1.
- the test item was applied to the monolayer of BALB/3T3 cells and was incubated at (37 ⁇ 1)°C in (5 ⁇ 1)% C02 atmosphere for 24 hours.
- a qualitative evaluation was performed observing cell culture by means of an inverted microscope, while a quantitative evaluation was performed using the Neutral Red Uptake method (NRU).
- the NRU is a method to measure cell viability using their capacity to incorporate and to bind a cellular viability dye, the Neutral Red.
- Qualitative evaluation After incubation the cells were observed under the light microscope to evaluate the biological reactions. After 24 hours of contact, in the cells treated with test sample no detectable zone around or under specimen has been observed (reactivity grade 0).
- DMEM Dulbecco's Modification of Eagle's Medium
- DPBS Dulbecco's Phosphate-Buffered Saline
- Pen-Strep Penicillin-Streptomycin
- FBS Foetal Bovine Serum
- WFJ Glacial acetic acid Neutral Red dye
- Ethanol Ethanol
- Trypsin EDTA Trypan Blue Common laboratory equipment Laminar flow filtered work area Biohazard Hood Inverted Microscope Diavert Microplate reader C02 incubator Orbital shaker Chronometer Refrigerator 6 mm Whatman inert filter paper Controls Dulbecco's Phosphate-Buffered Saline (DPBS, negative control)* Sodium Dodecyl Sulphate (SDS/SLS, positive control)* *deposed on a 6 mm Whatman inert filter paper EXPERIMENTAL DESIGN PLATE PREPARATION From a Mammal fibroblasts BAlB/3T3
- Test sample preparation As Requested by the Sponsor, the powder contained in the vial (80 mg) was suspended with 2 ml of WFI to reach the concentration of 40 mg/ml. 50 ⁇ L of the test sample were deposed on inert filter paper placed then in the middle of each well. [3 replicates] Negative control preparation The negative control was represented by 50 ⁇ L of DPBS deposed on an inert filter paper placed in the middle of each well. [3 replicates] Positive control preparation The positive control was represented by 50 ⁇ L of 0.4% solution of SOS /SLS deposed on an inert filter paper placed in the middle of each well.
- the absorbance of the resulting solution was measured at 540 nm in a microtiter plate reader OBSERVATIONS Qualitative evaluation
- the biological reactivity (cell degeneration and malformations) was evaluated after 24 hours of incubation with a scale ranging from 0 to 4, according to ISO 10993-5 as shown in the following table :
- Optical density was measured at 540 nm (Gen5 - Biotek).
- Percentages of cell viability was calculated according to the formula : INTERPRETATION OF RESULTS Qualitative evaluation The achievement of a numerical grade greater than 2 was considered a cytotoxic effect.
- Quantitative evaluation A cellular viability reduction >30% was considered a cytotoxic effect.
- Preliminary test A preliminary test was intended to determine the concentration of the test sample to be used in the main test. For the topical induction phase, the highest concentration that causes mild to moderate erythema but does not otherwise adversely affect the animal was selected. For the challenge phase, the highest concentration that produces no erythema was selected. To select the sample dilution, four occlusive patches with 0.5 ml of the undiluted sample and diluted sample (90%, 80% and 70% in Sodium Chloride Injection) were applied to the dorsum of three additional animals. The dressing was left in place for 24 hours.
- Animal selection Animals used in the study were randomly selected among the ones available at the time of the study. Caging The animals were caged according to internal procedure. The housing room was lighted with fluorescent lamps and maintained with cycles of 12 hours of light and 12 hours of dark. Room temperature and humidity were regulated by a conditioning plant and were daily monitored. Continuous recordings of the housing conditions are being retained in Eurofins Biolab S.r.l. files. Cleaning and disinfection The cages and the housing room were cleaned before animal accommodation, then periodically cleaned and disinfected. Feeding Animals have been fed with standard pellet complete diet supplied by the authorized breeder.
- the highest concentration that produces no erythema was selected.
- SAMPLE PREPARATION The powder contained in the vial (80mg) was suspended in 2ml of W FI (Batch: 1902401) to reach the concentration of 40 mg/ml before use.
- EXPERIMENTAL DESIGN consisted of one group of 10 treated animals (group 1) and one group of 5 control animals (group 2). The animals were allocated into groups as follows: 2 At maximum 10 animals for each cage; cages have been identified via a tag TREATMENT The main test consisted of an induction phase and a challenge phase. Skin preparation About 7 hours before intradermal injection and about 23 hours before topical application of induction phase, fur was removed by shaving a 50 cm 2 wide area on the interscapular region of the animals. About 23 hours before the challenge phase the fur on both flanks of animals was removed.
- the intensity of erythema and/or oedema was evaluated according to the following scale (Magnusson and Kligman -18010993-10): INTERPRETATION OF RESULTS Generally, Magnusson and Kligman grades of 1 or greater in the test group indicate sensitizatio, provided grades of less than 1 are seen in control animals. If grades of 1 or greater are noticed in control animals, the reactions of test animals which exceed the most severe reaction in control animals are presumed to be due to sensitization. If the response is equivocal, re-challenge is recommended to confirm the results from the first challenge. The outcome of the test is presented as the frequency of positive challenge results in the treated and control animals.
- Body weight has been measured immediately before the injections, 24, 48 and 72 hours after the injections. Clinical symptoms In none of the treated and control animals' toxic signs or symptoms were observed. Mortality In none of the treated and control animals' mortality was observed. Weight increase No weight loss was recorded in any treated and control animal. On the basis of the results, interpreted according to ISO 10993-11:2017, the test item "BIO ACTIVE COLL" DOESN'T CAUSE toxic symptoms and SATISFIES the requirements of the test. TEST METHOD Justification of assay system Mouse has been used for this test because of the recommendation of ISO 10993-11- current edition. Caging The animals were caged according to internal procedure. The housing room was lit with fluorescent lamps 12 hours per day. Room temperature and humidity were regulated by a conditioning plan and were monitored continuously.
- EXPERIMENTAL DESIGN The experimental design consisted of 2 groups (1 treated and 1 control) each consisting of five male mice. The animals were subdivided in groups as follow: I.P.: intra-peritoneal Sample preparation As Requested by the Sponsor, the powder contained in the vial (80mg) was suspended with 2ml of WFI to reach the concentration of 40 mg/ml before use. Treatment Animals have been treated with a single injection of test sample (treated groups) and Sodium Chloride Injection (control groups) with a dose of 50 ml/kg. Body weight measurement Body weight of all animals has been measured immediately before the injections, 24, 48 and 72 hours after the injections.
- OBSERVATIONS All animals have been observed after injection and after 4, 24, 48 and 72 hours. Eventual clinical signs (time of onset, degree and duration) and/or eventual mortality have been recorded for each animal. Animals were observed for the following systemic effects: tremors, hair bristling, diarrhoea, abdominal pain, sialorrhoea, depression state of sensorium, state of excitement, polypnoea, hypopnoea, tachycardia, cyanosis, ataxia, convulsions, nose- bleeding. Other clinical signs described in ISO 10993-11, Annex C, (if present) has been recorded.
- test conditions are satisfied if none of the animals treated with the sample show significantly greater biological reactivity than control group. If any of the animals treated with the sample show slight signs of biological reactivity, and no more than one animal show gross symptoms of biological reactivity or die, the test must be repeated using groups of 10 mice. The conditions of the repeated test are satisfied if during observation period none of the animals treated with the sample show a biological reactivity greater than the animals treated with the control. If two or more mice die, if two or more mice show abnormal symptoms as convulsions or weakness or if the weight loss is greater than 10% in 3 or more animals the test substance does not satisfy the requirements of the test.
- ISO 10993-11:2017 describe the approach to be used for the evaluation of pyrogenicity of medical devices and states that “Pyrogenicity is the ability of a chemical agent or other substance to produce a febrile response. Pyrogenic responses may be material- mediated, endotoxin-mediated, or mediated by other substances, such as components of gram-positive bacteria and fungi”. As defined by ISO 10993-11:2017, for detection of material-mediated pyrogenicity on medical devices, the rabbit pyrogen test is recommended. The test shall be performed according to the method described in the United States Pharmacopoeia, the European Pharmacopoeia and the Japanese Pharmacopoeia.
- Bio Active Coll - TYPE1 PROFIL is intended to be used for the regeneration of the dermic and sub-dermic cutaneous tissue.
- the device is intended to be applied topically on the skin.
- the device consists in a water-soluble sterile powder made of collagen (Type I) from equine origin.
- the device shall be dissolved in water for injection or saline solution, at the concentration of 2040mg/ml.
- the device is supplied in vials containing 80 mg of powder Assessment of material mediated pyrogenicity for “Bio Active Coll” According to USP ⁇ 151>, the test should be performed using an extract of the device, which should be injected into an ear vein of 3 rabbits.
- test results are not useful to characterize the potential risk of material-mediated pyrogenicity of the medical device.
- Annex G of ISO 10993-11:2017 states: “It is not necessary to test all new medical devices for in vivo pyrogenicity. However, materials containing substances that have previously elicited a pyrogenic response, and/or new chemical entities where the pyrogenic potential is unknown should be evaluated for material-mediated pyrogenicity”.
- BIO ACTIVE COLL was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus (tk+/-) using the L5178Y cell line.
- STE short-term experiments
- the solved test item was incubated with cells for 4 hours.
- the STEs were performed independently: with (STE(+)) and without (STE(-)) metabolic activation.
- LTE long-term experiment
- the LTE was performed without metabolic activation.
- the solved test item was investigated at the following concentrations: STE(-): 0.25, 0.50, 1, 3 and 5 mg/mL STE(+): 0.25, 0.50, 1, 3 and 5 mg/mL LTE: 0.25, 0.50, 1, 3 and 5 mg/mL No precipitation of the solved test item was noted in the experiments. No growth inhibition was observed in STE(-). Growth inhibition was observed in STE(+). The relative total growth (RTG) was 69%, for the highest concentration (5 mg/mL) evaluated. Also, growth inhibition was observed in LTE. The relative total growth (RTG) was 26% for highest concentration (5 mg/mL) evaluated. No biologically relevant increase of mutants was found in any experiment.
- the global evaluation factor (GEF) was not exceeded by the induced mutant frequency.
- EMS, MMS, and B[a]P were used as positive controls and demonstrated distinct and biologically relevant responses in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potentially clastogenic effects.
- Summary Conclusion In conclusion, in this in vitro Mammalian Cell Gene Mutation Assay (Thymidine Kinase Locus/tk+/-) in L5178Y Mouse Lymphoma Cells, under the experimental conditions reported, the test item BIO ACTIVE COLL is considered to be non- mutagenic.
- Mammalian cell culture systems are used to detect mutations induced by chemical substances. This in vitro experiment is conducted to assess the potential of the test item to induce gene mutations by means of a thymidine kinase (tk) assay using the mouse lymphoma cell line L5178Y.
- tk thymidine kinase
- the thymidine kinase assay detects base pair mutations, frameshift mutations, small, large and non-lethal deletions and rearrangements of the relevant chromosomes.
- the kinase catalyses the reaction of thymidine and ATP to form TMP (thymidine 5'- mono-phosphate) and ADP. However, it also phosphorylates the pyridine analogue triflurothymidine (TFT) to its cytostatic and cytotoxic trifluoro-thymidine- monophosphate derivative. By inactivating the functional allele of tk+/-, cells are resistant to TFT. Thus, mutant cells (tk-/-) are capable of proliferation in the presence of TFT, whereas normal cells (tk +/-) are not.
- TFT pyridine analogue triflurothymidine
- Cells in suspension are exposed to the test chemical, both with and without an exogenous source of metabolic activation (MA), for a suitable period of time, and then sub-cultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection.
- MA metabolic activation
- Cytotoxicity is determined by relative total growth (RTG).
- RTG relative total growth
- test item identity was inspected upon delivery at the test facility (e.g. test item name, batch no., and additional data were compared with the label) based on the following specifications provided by the sponsor.
- Safety Precautions The routine hygienic procedures w'ere sufficient to assure personnel health and safety.
- the preparation was carried out in compliance to ISO 10993-3: 2014 “Tests for genotoxicity, carcinogenicity and reproductive toxicity”:
- test sample be dissolved/suspended in an appropriate solvent compatible with the test system?
- the solvent was compatible with the survival of the cells and the S9 activity. Controls
- the L5178Y tk +/- 3.7.2C cell line has been successfully used in in vitro experiments for many years. These cells are characterised by their high proliferation rate (10-12 h doubling time of the Eurofms Kunststoff stock cultures) and their cloning efficiency, typically more than 50%. The cells obtain a near diploid karyotype (40 ⁇ 2 chromosomes) and are heterozygous at the thymidine kinase (tk) locus.
- tk -/ - cells can be eliminated by culturing in RPMI 1640 supplemented with: 9.0 ⁇ g/mL, hypoxanthine, 15.0 ⁇ g/mL thymidine, 22.5 ⁇ g/mL glycine, 0.1 ⁇ g/mL methotrexate.
- the cells are resuspended in medium without methotrexate, but with thymidine, hypoxanthine, and glycine for 1-3 days.
- Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and sub-cultured on demand.
- Post-Mitochondrial Fraction (S9) Substances may only develop mutagenic potential when they are metabolised by the mammalian organism.
- the cell line L5178Y tk +/ - 3.7.2C has an inadequate endogenous metabolic capacity and therefore an exogenous MA system is necessary .
- the most commonly used system is a co-factor- supplemented post-mitochondrial fraction (S9) prepared from livers of rodents.
- the S9 fraction was prepared at Eurofms Kunststoff.
- Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and ⁇ -naphthoflavone (100 mg/kg bw) for three consecutive days by oral route. The preparation was performed according to Ames et ah.
- a stock of the supernatant containing the S9 fraction was frozen in aliquots of 2 and 4 mL and stored at ⁇ -75 °C.
- the protein concentration in the S9 fraction was 35 mg/mL (Lot: 191121).
- the S9 mix preparation was performed according to Ames et al..
- S9 fraction was thawed and mixed with a co-factor solution to result in a final protein concentration of 0.75 mg/mL in the cultures.
- concentrations were achieved: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 5 mM NADP.
- S9 mix was stored on ice.
- Criteria to determine the highest concentration were cytotoxicity, solubility in the test system, and changes in pH or osmolality. Cytotoxicity was determined with and without m etabolicactivation .
- the solved test item was removed by centrifugation (200xg, 7min)and the cells were washed twice with PBS. Subsequently the cells were suspended in 30 mL complete culture medium and incubated for an expression and growth period of two days in total at 37 °C in 5% CO2/95% humidified air. The cell density was determined each day and, if necessary, adjusted to 3x10 5 cells/mL. For the LTE, 5x10 6 cells were suspended in 25 mL RPMI medium with 7.5% horse serum (75 cm 2 flasks) and exposed to designated concentrations of the solved test item in the absence of MA. After 24 h, the solved test item was removed by centrifugation
- Relative suspension growth (RSG) and RTG (RTG [RSG x RCE] / 100) of the treated cell cultures were calculated according to the method of Clive and Specter. Additionally, cultures were seeded in selective medium. Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in 200 ⁇ L selective medium with TFT. The plates were scored after an incubation period of 11 to 14 days at 37 °C in 5% CO2/95% humidified air.
- the MF was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT.
- the MF is usually expressed as “mutants per 10 6 viable cells”
- Suspension growth (SG) of the cell cultures reflects the number of times the cell number increases from the starting cell density.
- SG Suspension growth
- the GEF is defined as the mean of the negative/vehicle MF plus one standard deviation. Applying this definition to the collected data, the GEF is 126 mutants per 10 6 cells for the microwell method.
- the size of the colonies was characterised as follows: Small colonies approximately ⁇ 1 ⁇ 4 of well diameter. Large colonies approximately > 1 ⁇ 4 of well diameter. Size is the key factor and morphology should be secondary.
- the selective agent was obtained from Sigma-AIdrich (Lot No.: BCCD1996). Data recording
- the generated data were recorded on the experimental design protocol.
- the results are presented in tabular form, including experimental groups with the test item, negative and positive controls.
- Individual colony counts for the treated and control groups are presented for both mutation induction and survival. Small colonies and large colonies were determined for the relevant test groups.
- the non-parametric Mann- Whitney test was applied to the mutation data to determine if there are statistical differences between the mutant frequencies of the treated test groups compared to the negative/solvent controls.
- a mutation assay is considered acceptable if it meets the criteria defined in current international guidelines and the current recommendations of the IWGT . At least three out of four 96-well plates from the TFT selection experiment are analy sable.
- the cloning efficiency of the negative or solvent controls is in the range 65%- 120%
- the spontaneous MF in the negative or solvent controls is in the range 50- 170 mutants per 10 6 cells.
- the cell number of the negative/solvent controls undergo 8-32-fold increase during a two-day growth period (STE) or 32-180-fold increase during a three-day growth period (LTE).
- the positive controls (MMS and B[a]P) for clastogenicity produce an induced
- the RTG must be greater than or equal to 10%.
- test item is considered mutagenic if the following criteria are met:
- the induced MF meets or exceeds the GEF .
- the RTG must be greater than or equal to 10%.
- a test item is considered negative if the induced MF is below the GEF or the trend of the test is negative.
- test item BIO ACTIVE COLL was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus (tk +/- using the L5178Y cell line.
- tk +/- mouse lymphoma thymidine kinase locus
- STE short-term experiments
- the solved test item was investigated at the following concentrations:
- the measured pH value of the solved test item was within the physiological range. No precipitation of the solved test item was noted in the experiments.
- the global evaluation factor (GEF) was not exceeded by the induced mutant frequency.
- the GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation, data are gathered from ten laboratories.
- the GEF was defined to be 126 mutants/10 6 cells.
- Criterion for mutagenicity is the extension of the GEF by the induced mutant frequency as well as a concentration-dependent increase in mutant frequency.
- the positive controls EMS (200 and 300 ⁇ g/mL), MMS (8 and 10 ⁇ g/mL) andB[a]P (3.5 ⁇ g/mL) showed distinct responses in mutation frequency, thus demonstrating the ability of the test system to detect potentially mutagenic effects.
- BIO ACTIVE COLL In order to investigate the potential of BIO ACTIVE COLL for its ability to induce gene mutations the plate incorporation test was performed with the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM101).
- BIO ACTIVE COLL did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
- BIO ACTIVE COLL is considered to be non-mutagenic in this bacterial reverse mutation assay.
- Bacterial reverse mutation assays use amino acid requiring strains of Salmonella typhimurium and Escherichia coli (E. coli) to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs.
- the principle of these bacterial reversion assays is that they detect mutations which functionally reverse mutations present in the tester strains and restore the capability to synthesize an essential amino acid.
- the S. typhimurium histidine (his) reversion system and the E. coli tryptophan (trp) reversion systems measure his- ->his + reversions and trp trp + .
- the S. typhimurium strains are constructed to differentiate between base pair (TA100, TA1535) and frameshift (TA98, TA1537) mutations.
- the E. coli strain detects only base substitution mutagens.
- the bacteria are exposed to the test item with and without metabolic activation and plated on selective medium. After a suitable period of incubation, revertant colonies are counted. At least five different concentrations of the test item are tested with approximately half log (i ,e. V 10) intervals between test points for an initial test. Narrower spacing between dose levels may be appropriate when a dose response is investigated. For soluble, non- toxic test compounds the recommended maximum test concentration is 5 mg/plate or 5 ⁇ L/plate.
- test item identity was inspected upon delivery at the test facility (e.g. test item name, batch no. and additional data were compared with the label) based on the following specifications provided by the sponsor. The following listed information applies to the sample as received.
- test item was prepared in compliance to ISO 10993-3: 2014 “Tests for genotoxicity, carcinogenicity' and reproductive toxicity” .
- the sample preparation should follow the decision tree in the Figure 3. This figure diagrams the decision process used to select the extraction method (method A, B or C).
- test sample can be dissolved or suspended in an appropriate solvent within the test system, the test sample can be applied directly to the test system (Method A) at a maximum concentration of 5 mg/niL (in vitro mammalian test system) or 5 mg/plate (bacterial reverse mutation assay).
- test item can be dissolved (see Eurofms Kunststoff Study 150459 “In vitro Cytotoxicity Assay: Evaluation of Materials for Medical Devices by Extraction Method and XTT Dye with BIO ACTIVE COLL”) Method A was chosen.
- the test item was dissolved in A. dest, processed by ultrasound for 5 min at 37 °C and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.
- Negative controls (A. dest., Eurofms Kunststoff, Lot No. 220218, 220304) were treated in the same way as all dose groups. Positive Controls
- Tester Strains S. typhimurium: TA100, TA1535
- Tester Strains S. typhimurium: TA98, TA1537 Name: 4-NOPD; 4-nitro-o-phenyl ene-diamine
- Tester Strain E. coli WP2 uvrA (pKMIOl)
- Tester strains TA98, TA1535 and E. coli were obtained from MOLTOX, INC., NC 28607, USA.
- Tester strains TA100 and TA1537 were obtained from Xenometrix AG, Switzerland. They were stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen. All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium.
- the other mutation i s a deletion of the uvrB gene coding for a protein of the DNA nucleotide excision repair system resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chl) and biotin (bio) genes (bacteria require biotin for growth).
- the tester strains TA98, TA100 and E. coli contain the R-factor plasmid, pkM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains. pkM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system which is normally present in these organisms.
- the tester strain E. coli WP2 uvrA (pKM101) carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagens which substitute one base for another. Additionally, the strain is deficient in the DNA nucleotide excision repair system. The properties of the S. typhimurium and E.
- the S. typhimurium medium contains per litre of purified water:
- the E. coli medium (Luria Bertani) contains per litre of purified water:
- the Vogel-Bonner Medium E agar plates with 2% glucose used in the Ames test were prepared by Eurofins Kunststoff or provided by an appropriate supplier. Quality controls were performed.
- Sterilisation was performed for 20 min at 121 °C in an autoclave.
- the overlay agar contains per litre of purified water:
- Sterilisation was performed for 20 min at 121 °C in an autoclave.
- S9 Homogenate The S9 liver microsomal fraction was prepared at Eurofms Kunststoff. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and b-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.
- a stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at ⁇ -75 °C.
- the S9 mix preparation was performed according to Ames et al.
- the S9 mix substitution buffer was used in the study as a replacement for S9 mix, without metabolic activation (-S9).
- Phosphate-buffer (0.2 M) contains per litre of purified water:
- This 0.2 M phosphate-buffer was mixed with 0.15 M KCl solution (sterile) in the following proportion:
- the toxicity of the test item was determined with tester strains TA98 and TA100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment (plate incorporation test).
- Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ⁇ 0.5 in relation to the solvent control.
- the test item was tested in the pre-experiment with the following concentrations:
- test item concentrations to be applied in the main experiment wore chosen according to the results of the pre-experiment. 5000 ⁇ g/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. The experiment was performed with the following concentrations: 31.6, 100, 316, 1000, 2500 and 5000 gg/plate
- test solution 100 ⁇ L test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control)
- coli WP2 uvrA (pKM101)) - the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (January – December 2020 for all tester strains)): - corresponding background growth on negative control, solvent control and test plates is observed - the positive controls show a distinct enhancement of revertant rates over the control plate - at least five different concentrations of each tester strain are analysable. Evaluation of Mutagenicity The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
- a test item is considered as mutagenic if: - a clear and dose-related increase in the number of revertants occurs and/or - a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
- a biologically relevant increase is described as follows: if in tester strains TA98, TA100 and E. coli the number of reversions is at least twice as high if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
- the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
- Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ⁇ 0.5 in relation to the solvent control.
- test item BIO ACTIVE COLL was investigated for its potential to induce gene mutations according to the plate incorporation test using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101).
- BIO ACTIVE COLL did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
- BIO ACTIVE COLL is considered to be non-mutagenic in this bacterial reverse mutation assay.
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Title |
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DATABASE GNPD [online] MINTEL; 12 May 2021 (2021-05-12), ANONYMOUS: "Hydrolyzed Collagen Sachets", XP055902997, retrieved from https://www.gnpd.com/sinatra/recordpage/8701009/ Database accession no. 8701009 * |
DATABASE GNPD [online] MINTEL; 27 April 2021 (2021-04-27), ANONYMOUS: "Original Hydrolyzed Collagen Powder", XP055902994, retrieved from https://www.gnpd.com/sinatra/recordpage/8657549/ Database accession no. 8657549 * |
LEÓN-LÓPEZ ARELY ET AL: "Hydrolyzed Collagen-Sources and Applications", vol. 24, no. 22, 7 November 2019 (2019-11-07), pages 4031, XP055903004, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC6891674/pdf/molecules-24-04031.pdf> DOI: 10.3390/molecules24224031 * |
MATHEW-STEINER SHOMITA S. ET AL: "Collagen in Wound Healing", vol. 8, no. 5, 1 January 2021 (2021-01-01), pages 63, XP055903006, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC8151502/pdf/bioengineering-08-00063.pdf> DOI: 10.3390/bioengineering8050063 * |
MOSKOWITZ R W ED - BREEDVELD FERDINAND CHRISTOFFEL: "ROLE OF COLLAGEN HYDROLYSATE IN BONE AND JOINT DISEASE", SEMINARS IN ARTHRITIS AND RHEUMATISM, ELSEVIER, AMSTERDAM, NL, vol. 30, no. 2, 1 October 2000 (2000-10-01), pages 87 - 99, XP009021767, ISSN: 0049-0172, DOI: 10.1053/SARH.2000.9622 * |
SUGIHARA FUMIHITO ET AL: "Ingestion of bioactive collagen hydrolysates enhanced pressure ulcer healing in a randomized double-blind placebo-controlled clinical study", vol. 8, no. 1, 1 December 2018 (2018-12-01), pages 11403, XP055903007, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC6065362/pdf/41598_2018_Article_29831.pdf> DOI: 10.1038/s41598-018-29831-7 * |
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